1. Field of the Invention
The present invention concerns the novel biosynthetic genes for encoding the proteins responsible for producing the LL-F28249 compounds and the use thereof to make the active metabolites from the fermentation of Streptomyces cyaneogriseus subsp. noncyanogenus. The invention further concerns the genetic manipulation of the biosynthetic pathway to make active semisynthetic derivatives of the natural metabolites.
2. Description of the Related Art
All patents and publications cited in this specification are hereby incorporated by reference in their entirety.
Streptomyces are producers of a wide variety of commercially important secondary metabolites, including the majority of active antibiotics known as the β-lactams and the macrocyclic lactone compounds or macrolides. Because of the commercial importance of the secondary metabolites produced by Streptomyces, there has been considerable recent investment in the development of methods for molecular genetic manipulation of Streptomyces. Procedures have been developed for the introduction of genetic material by polyethylene glycol mediated transformation and by conjugal transfer from Escherichia coli. Vectors have been developed including high and low copy number vectors, integrative vectors, and E. coli-Streptomyces shuttle vectors. These methods for molecular genetic manipulation of Streptomyces have been summarized in D. A. Hopwood et al., Genetic Manipulation of Streptomyces. A Laboratory Manual, John Innes Foundation Press, Norwich, UK (1985). In many cases, the genes for the production of secondary metabolites are clustered in Streptomyces. Thus, the identification of a single gene in a biosynthetic gene cluster may lead to the identification of all of the genes responsible for the biosynthesis of the metabolite. This observation has proven to be tremendously valuable, and secondary metabolite biosynthetic gene clusters have been cloned by reverse genetics, complementation of blocked mutants, resistance and use of heterologous probes. Using these methods, nucleotide and predicted amino acid sequence data have been obtained for many macrolide biosynthetic gene clusters including those directing the synthesis of erythromycin (see S. Donadio et al., Science 252:675-679 (1991) and S. F. Haydock et al., Molecular and General Genetics 230:120-128 (1991)); rapamycin (see T. Schwecke et al., Proceedings of the National Academy of Sciences USA 92:7839-7843 (1995) and X. Ruan et al., Gene 203:1-9 (1997)); FK506 (H. Motamedi and A. Shafiee, European Journal of Biochemistry 256:528-534 (1998)); oleandomycin (D. G. Swan et al., Molecular and General Genetics 242:358-362 (1994)) and rifamycin (see P. R. August et al., Chemistry & Biology 5:69-79 (1998)). However, the complete biosynthetic gene cluster for the macrocyclic lactone compounds known as the LL-F28249 compounds has not yet been described in the art.
There are many reports that molecular genetic manipulations can be used to alter the course of polyketide biosynthesis (see S. Donadio et al., Science 252:675-679 (1991) and S. Donadio et al., Proceedings of the National Academy of Sciences USA 90: 7119-7123 (1993)). In those studies, erythromycin-related lactones were produced following manipulation of the 6-deoxyerythronolide B synthase (“DEBS”) gene cluster (the core polyketide synthase gene cluster responsible for erythromycin biosynthesis) such that either the module 4 enoylreductase or the module 5 ketoreductase domains were nonfunctional. Strains containing these variant DEBS gene clusters produced the expected erythromycin-related lactones. These pioneering studies have since been repeated and expanded upon, and the results of many such studies have been reviewed in the literature (see, for example, L. Katz and S. Donadio, Annual Reviews of Microbiology 47:875-912 (1993); C. R. Hutchinson and I. Fujii, Annual Reviews of Microbiology 49:201-238 (1995); D. A. Hopwood, Chemical Reviews 97:2465-2497 (1997); and C. W. Carreras and D. V. Santi, Current Opinions in Biotechnology 9:403-411 (1998)).
Data summarized in the literature suggest that the organization of catalytic domains in type I polyketide synthase (“PKS”) modules is conserved, and many highly conserved amino acid sequence motifs have also been described in those biosynthetic gene clusters. For example, the organization of the biosynthetic gene cluster of avermectin, which is produced by S. avermitilis, has been reported (see D. J. MacNeil et al., Gene 115:119-125 (1992) and D. J. MacNeil et al., Annals of the New York Academy of Sciences 721:123-132 (1994)); and partial nucleotide sequences of that biosynthetic gene cluster have been reported or are otherwise available. MacNeil and colleagues have also predicted the modular organization and reported a limited restriction endonuclease map of the wild-type S. cyaneogriseus (NRRL 15773) nemadectin biosynthetic gene cluster (see D. J. MacNeil et al., Annals of the New York Academy of Sciences 721:123-132 (1994)), but their restriction map was incomplete. Their analysis only indicated the presence of nine modular repeats of PKS function and required six overlapping clones to define the 75 kb region of the S. cyaneogriseus genome. MacNeil et al. did not complete the DNA sequencing of the whole biosynthetic gene cluster. Instead, the authors sequenced only the ends of selected cosmids. From the limited sequence information, they could only generate a very sketchy restriction endonuclease map. Further C-13 labeling studies have been conducted, and a mechanism for synthesis of the LL-F28249α compound from its constituent acyl units has been proposed (H. R. Tsou et al., Journal of Antibiotics (Tokyo) 42:398-406 (1989)).
The highly active LL-F28249 compounds, which are natural endectocidal agents widely used for treatment of nematode and arthropod parasites, including the control or prevention of helmintic, arthropod ectoparasitic and acaridal infections, are isolated from the fermentation broth of Streptomyces cyaneogriseus subsp. noncyanogenus (hereinafter referred to as “S. cyaneogriseus”). The series of anti-parasitic LL-F28249 compounds produced from S. cyaneogriseus are structurally similar to, but patentably distinct from, the well-characterized avermectins. U.S. Pat. No. 5,106,994 and its continuation U.S. Pat. No. 5,169,956 describe the preparation of the major and minor components, LL-F28249α-λ. The LL-F28249 family of compounds further includes, but is not limited to, the semisynthetic 23-oxo derivatives and 23-imino derivatives of LL-F28249α-λ, which are shown in U.S. Pat. No. 4,916,154. Moxidectin, chemically known as 23-(O-methyloxime)-LL-F28249α, is a particularly potent 23-imino derivative. Other examples of LL-F28249 derivatives include, but are not limited to, 23-(O-methyloxime)-5-(phenoxyacetoxy)-LL-F28249α, 23-(semicarbazone)-LL-F28249α and 23-(thiosemicarbazone)-LL-F28249α.
One of the major nemadectin metabolites, LL-F28249α (hereinafter referred to as “Fα”), is converted to the commercially important compound moxidectin using a four-step chemical process. The determination of the biosynthetic gene cluster of Fα, heretofore unknown, would be of great commercial significance. Not only would isolation of the gene be highly desirable to make the active Fα compound and other natural members of the LL-F28249 family of compounds, but also to prepare the commercially potent semisynthetic derivatives such as moxidectin more quickly and efficiently.
It is therefore an important object of the present invention to isolate and characterize the entire nucleotide sequence encoding the proteins responsible for producing the LL-F28249 compounds, preferably the LL-F28249α metabolite, and then to isolate and determine the function of the amino acid sequences comprising the biosynthesis proteins.
Another object is to provide a new process for isolating natural and semisynthetic derivatives directly from the fermentation broth of bioengineered strains of Streptomyces cyaneogriseus subsp. noncyanogenus. 
A further object is to provide a new method for the preparation of moxidectin in an efficient process with fewer steps than heretofore achievable.
Further purposes and objects of the present invention will appear as the specification proceeds.
The foregoing objects are accomplished by providing a new, purified and isolated nucleic acid molecule that encodes the proteins connected with the entire biosynthetic pathway for producing the LL-F28249 compounds.